Diagnostic preparation for the detection of acetylmethylcarbinol



Dec. 19, 1967 G. I EVANS ETAL 3,359,180

DIAGNOSTIC PREPARATION FOR THE DETECTION OF ACETYLMETHYLCARBINOLI FiledApril 4, 1967 BARRIER ZONE 4.

REAGENT ZONE 3. (SOLUTION A) 7 INVENTOR. GEORGE L. EVANS CHARLES I.HELLER BENJAMIN s. SCHWARTZ ATTORNEY United States Patent 3,359,180DIAGNOSTIC PREPARATION FOR THE DETEC- TION 0F ACETYLMETI-IYLCARBINOLGeorge L. Evans, Hopatcong, Charles I. Heller, Fort Lee, and Benjamin S.Schwartz, Livingston, N.J., assignors to Warner-Lambert PharmaceuticalCompany, Morris Plains, N.J., a corporation of Delaware Filed Apr. 4,1967, Ser. No. 628,385 11 Claims. (Cl. 195-1035) ABSTRACT OF THEDISCLOSURE A method for differentiating and identifyingacetylmethylcarbinol (AMC) producing Enterobacteriaceae from non-AMCproducing Enterobacteriaceae which utilizes a bibulous strip containinga medium zone capable of supporting the growth of Enterbacteriaceae,separated by a hydrophobic barrier from a reaction zone containing anacid addition salt of l-arginine and an alkali metal salt of ot-naphtholsulfonic acid. The strip is inserted into a tube containing an unknownorganism isolated in pure culture with the medium zone of the stripimmersed in the organism suspension. After incubation, an alkali metalhydroxide solution is added to the test tube. A positive test isindicated by the production of a red color in the reaction zone of thebibulous test strip.

BACKGROUND OF THE INVENTION This application is a continuation in partof applicants copending U.S. patent application Ser. No. 427,272, filedon J an. 22, 1965, which describes a diagnostic preparation for therapid detection of acetylmethylcarbinol (AMC) produced by certainmicro-organisms. The diagnostic preparation described in Serial No.427,272 utilizes a bibulous strip with a medium zone consisting of BrainHeart Infusion Broth, trypticase and D-glucose separated by a hydrophobibarrier from a reaction zone consisting of l-arginine and ot-naphthol.The diagnostic test strip prepared according to this formulationprovided satisfactory results When used to detect the presence ofcertain AMC producing Enterobacteriaceae. However, it was subsequentlyfound that the test strips prepared with these ingredi'ents exhibited arelatively short shelf life. The anaphthol in the reagent zone wassomewhat unstable as it had a tendency to sublime, thereby spreading toall of the remaining portions of the strip. Also, it was found that thea-naphthol was soluble in the organic solvent in which the hydrophobicbarrier material is dissolved, thus it was .possible for the u-naphtholto spread to the remaining portions of the test strip. It was alsodiscovered that the a-naphthol was toxic to the organisms which weregrowing in the medium zone of the strip; therefore, when the u-naphtholspread to the medium zone of the bibulous strip, it tended to destroythe usefulness of this test procedure.

The production and the subsequent detection of acetyl-- methylcarbinol(AMC) from glycolysis products of certain organisms of theEnterobacteriaceae forms the basis of the Voges-Proskauer or V-P test.This test is a wellknown laboratory diagnostic method for thedifferentiation and identification of the various organisms Which belongto this group. For example, two organisms of this coliform group, i.e.,Aerobacter and Klebsiella, produce acetylmethylcarbinol and on thisbasis may be distinguished from Escherichia coli and E. frezma'iz',which do not. However, to carry out this classical test or its moremodern adaptations requires the use of incubation periods of a durationtoo long for rapid identification of the organisms.

3,359,180 Patented Dec. 19, 1967 Thus, one of the preferred methods fordetecting acetylmethylcarbinol is the Barritt modification (J. Path. 42:441-454, 1936). In this method the unknown organism isolated in pureculture from a specimen such as stool, blood or urine is grown in aglucose-peptone-phosphate buffer broth for 48 hours. To 1 ml. of themedium is then added 0.2 ml. 40% potassium hydroxide and 0.6 ml. 5%naphthol in ethanol. The development of a pink to red color in 2-5 min.,becoming crimson in 30 minutes to 4 hours indicates the presence ofacetylmethylcarbinol (AMC).

Another modificatioin of the V-P test is the Benjaminson method (M. A.Benjaminson, B. C. deGunsman, and A. I. Weil, I. Bact. 87,234-235,1964). In this test, isolates grown on triple sugar iron (TSI) agarslants are suspended in 0.5 ml. of 0.5% aqueous creatine solution andthen naphthol and 40% KOH are added. A pink to red color which developsin 5 minutes is positive for AMC.

It is obvious that there are many disadvantages to these methods. In theBarritt modification, time of incubation is dependent on the volume ofreagents and is critical. In addition, pure cultures must be grown inspecialized media following primary isolation of the culture. In themethod of Benjaminson et al., organisms can be taken directly from a TSIagar slant only if acid has been produced on the slant. The use oforganisms from a TSI slant is considered advantageous since this mediais usually incorporated into the scheme for separation of theEnterobacteriaceae. However, if acid is not produced on the slant, thetesting of these non-lactose fermentors becomes impractical. Hence, thetest is limited in practice to the Aerobacter-Klebsiella group to theexclusion of other AMC producers.

In all bacterial infections the rapid and accurate identification of theinfectious agent is of paramount importance so that proper therapy canbe promptly initiated. The above-described tests are obviouslyimpractical when rapid diagnosis is essential. In addition, thenecessity for the preparation of the various specific reagents requiredfor the tests further hampers the ability to achieve rapididentification. Also, the a-naphthol used in the tests outlined above isnot a stable reagent; therefore, it is neces sary that this reagent beprepared frequently and in small quantities.

SUMMARY OF THE INVENTION This invention relates to a novel method forthe differentiation and identification of acetylmethylcarbinol producingEnterobacteriaceae utilizing a bibulous strip containing a medium zoneand a reagent zone. The strip is inserted into a tube containing unknownorganisms isolated in pure culture; the tube containing the strip isthen incubated, after which an alkali metal hydroxide solution is addedto the tube With a resultant color change in the reaction zone of thestrip indicating the presence of AMC producing Enterobacteriaceae.

DESCRIPTION OF THE DRAWING The drawing shows a diagramaticrepresentation of the bibulous test strip with the medium zone and thereagent zone separated thereon by a hydrophobic barrier zone with asecond hydrophobic barrier zone contiguous to the reagent zone on theopposite side of the reagent zone from the first barrier zone.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The description of thisinvention may be more easily understood by reference to FIGURE I, whichis a diagramatic representation of the invention. The figures inparenthesis appearing herein relate to FIGURE I.

This invention relates to an improved diagnostic method fordifferentiating and identifying acetylmeth-ylcarbinol (AMC) producingEnterobacteriaceae from non- AMC producing Enterobacteriaceae whichutilizes a bibulous strip with a medium zone containing a medium capableof supporting the growth of Enterobacteriaceae separated by ahydrophobic barrier zone from a reagent zone which contains (a) acompound containing the guanidino group and (b) an alkali metal salt ofot-naphthol sulfonic acid.

The concentrations expressed herein are all parts by weight of the totalvolume of the aqueous solutions applied to the bibulous strip materialcommonly expressed as a weight/volume solution.

The bibulous test strip of the present invention is prepared byimpregnating one zone of the strip, hereinafter called reagent zone 3,with an aqueous reagent system which contains (a) a compound containingthe guanidino group, such as l-arginine or its mono, di, or tri acidaddition salts, agmatine, creatine, etc., wherein the acid additionsalts of l-arginine are those formed with strong mineral acids such ashydrochloric or nitric, and (b) an aqueous solution of an alkali metalsalt of a-n-aphthol sulfonic acid such as:

l-naphthol-S-sulfonic acid sodium salt, l-naphthol-S-sulfonic acidsodium salt, l-naphthol-Z-sulfonic acid potassium salt,1-naphthol-3,6-disulfonic acid disodium alt, and 1-naphthol-4-sulfonicacid sodium salt.

The alkali metal salts of naphthol sulfonic acid are soluble in waterbut insoluble in the organic vehicles in which the hydrophobic barriermaterial is dissolved. When applied to the test strip, these salts arestable and do not sublime from one zone of the test strip to another.

The reagent system contains an aqueous solution of from about 2% toabout 3% w./v. of a compound containing the guanidino group with fromabout 3% to about 4% w./v. of an alkali metal salt of ot-naphtholsulfonic acid added thereto. The reagent system, which preferably ismade up of the sodium salt of 1-napthol-8- sulfonic acid and l-argininemonohydrochloride, is applied to the reagent zone 3 of the strip inexcess amounts. The ingredients of the reagent zone function as colordevelopers when they come into contact with AMC. Therefore, the amountof these two ingredients which is necessary for the reaction zone of thetest strip is a function of the amount of AMC produced by themicro-organisms. Consequently, the amount of these two ingredients inthe reagent zone is not critical, so long as they are present in excessamounts. The preferred concentration of the ingredients in the reagentzone of the strip is 0.3 mg. of 1-naphthol-8-sulfonic acid sodium saltper strip and 0.2 mg. of l-arginine per strip. The ingredients in thereagent system may be applied in the relative proportions of about 1 toabout 5 parts of an alkali metal salt of a-naphthol sulfonic acid toabout 1 part by weight of a compound containing the guanidino group. Thepreferred ratio in which these ingredients are applied is about 1.2parts of an alkali metal salt of a-naphthol sulfonic acid, preferably1-naphthol-8-sulfonic acid sodium salt to about 1 part by weight of acompound containing the guanidino group, preferably l-argininemonohydrochloride. These ingredients may be applied to the bibulousstrip with a syringe microbiuret No. S 1/ 4T (Model No. SBZ),Micro-Meter Instrument Co., Cleveland, Ohio.

Impregnated in another zone of the strip, hereinafter called the mediumzone 1, is an aqueous nutrient medium containing ingredients capable ofsupporting the growth of Enterobacteriaceae, which ingredients may be apeptone-dextrose-potassium phosphate mixture, with D-glucose and sodiumpyruvate optionally included therein as desirable adjuncts which promotethe rapid production of AMC. In the practice of this invention, thepeptonedextrose,potassium phosphate mixture is preferably obtained in aprepared form known as Bacto MR-VP Medium in which thepeptone-deXtrose-potassium phosphate is present in a ratio of 7 partspeptone-S parts dextrose-5 parts potassium phosphate.

The concentration of the peptone-dextrose-phosphate mixture in thenutrient medium with which the medium zone 1 is impregnated is notcritical and may vary from about 5% to about 25% by weight of the totalweight of the nutrient medium. Where the optional ingredients D- glucoseand sodium pyruvate are in the nutrient medium with which the mediumzone 1 is impregnated, they may be included in a concentration varyingfrom 5% to 25 by weight of the total weight of the nutrient medium;however, it is preferred that the D-glucose be maintained at aconcentration of about 20% to 25% by weight of the nutrient medium togive optimum results.

The reagent zone 3 and the medium zone 1 are separated by a barrier zone2 containing hydrophobic barrier material. Another barrier zone 4containing hydrophobic barrier material is contiguous to reagent zone 3and on the opposite side of reagent zone 3 from zone 2.

The hydrophobic barrier zone 2 must be capable of isolating theingredients of the medium zone 1 from the ingredients of the reagentzone 3 during the incubation period hereinafter described. Barrier zone4 is preferably present in order to prevent excessive migration of thereagent and diffusing of the color formed in a positive test. Theingredients in barrier zones 2 and 4 must be microbiologically inertwith respect to the Enterobacteriaceae. Any substance which will form awaterproof barrier of this type may be used. Suitable materials includewaxes, lacquers, and a colorless acrylic resin composition known asKrylon 150 Crystal Clear. The Krylon material is particularly preferred.It is supplied in a toluene vehicle and may be diluted for ease ofapplication with additional toluene or other hydrocarbon thinners, suchas ethyl, methyl or propyl alcohol U.S.P. It has been found that barriersolution made from about 75% to 100% v./v. Krylon and 0 to 25% v./v.diluent is suitable. A particularly preferred combination is preparedfrom v./v. Krylon with 15% v./ v. ethyl alcohol U.S.P.

Bibulous materials which can be employed as the strip carriers are thosematerials which by means of capillary action are able to draw a liquidupward and materials such as filter paper,.felt, porous ceramic strips,woven or matted glass fiber and the like are suitable.

The bibulous material is normally cut into narrow strips to facilitatepackaging in small bottles or other containers from which they may bedispensed as needed.

In use, the end of the bibulous material impregnated with the nutrientmedium, the medium zone 1, is inserted into a suspension of a pureculture of unknown bacteria contained in a test tube, and while in thisposition in the tube, both the tube and strip are incubated together at37 C. for about 4 hours. The suspension of bacteria need not have beengrown in special media or incubated for long duration for this testalthough cells grown in a medium containing relatively highconcentrations of glucose give a better reaction. After incubation, afew drops of an aqueous alkali metal hydroxide are added to the tubetaking care not to wet the reagent zone. The concentration of the alkalimetal hydroxide solution is preferably 40% although greater amounts ofmore dilute solutions can be used.

The tube contents are then gently mixed after which the hydroxidetreated material is allowed to come in contact with the reagent zone. Apositive test for the presence of AMC is indicated by a magenta cherryred color which is quite prominent in the reagent zone. This cherry redcolor appears in about 5 to 30 minutes.

The advantages and convenience of the above-described invention arequite outstanding in view of the fact that the test for AMC can becarried out under conditions employing a very much shorter incubationtime then previously considered necessary. In addition, this test hasalso been found to be more sensitive for detecting the AMC production ofcertain bacteria than conventional techniques.

The following examples are included in order to further illustrate theinvention:

Example 1 (A) Preparation of reagent system (Solution A).-- 2. 6 gramsof l-arginine is dissolved in about 100 ml. of water. To this is addedwith stirring of 3.3 grams l-naphthol-S-sulfonic acid, sodium salt.

(B) Preparation of nutrient medium (Solution B). 17 grams of dehydratedBacto MR-VP Medium, grams of sodium pyruvate, 37.5 grams of D-glucoseare dissolved together in 150 ml. of distilled water with the aid ofgentle heating until the solution is complete.

(C) Application to bibulous material.In perparing the test strip theinitial step is the application of two barrier zones 2 and 4 which actto prevent the migration of aqueous solutions which are subsequentlyapplied. These barrier zones are applied to a suitable filter paper suchas Eaton-Dykman #623 by employing a solution of a lacquer which producesa water-impervious barrier. After allowing the lacquer solution to dry,the reagent zone 3, consisting of Solution A, of Example 1, and themedium zone 1, consisting of Solution B, of Example 1, as describedabove are then applied to the paper. The zones thus formed are thenallowed to dry. The filter paper is then cut into strips.

ExampleZ USE OF THE REAGENT STRIPS l to 2 loopfuls of an unknownbacterium isolated in pure culture, which was previously grown on anagar slant, is suspended in 0.3 ml. of isotonic saline. A test stripprepared according to Example 1 is placed in the tube with the mediumzone 1 immersed in the suspension of the organism. The test strip andthe organism suspension are incubated at 37 C. for about 4 hours. Afterthe incubation period, several drops of potassium hydroxide are added tothe tube, taking care to avoid Wetting the reagent zone 3. The tube isgently agitated and the suspension is then allowed to come in contactwith the reagent zone 3. A positive test for the presence ofacetylmethylcarbinol is indicated by the formation of a pink to redcolor in the reagent zone.

Test strips, prepared as described in Example 1 and used in the mannerdescribed in Example 2, were compared with conventional testing methodsused to detect the presence of acetyhnethylcarbinol. The resultsobtained are shown in Table 1:

TABLE 1.-CONVENTIONAL VOGES-PROSKAUER AND PAPER STRIP TEST REACTIONSAMONG REPRESEN- TATIVE GENERA AND SPECIES IN THE ENTEROBAC- TERIACEAE Itis understood that the foregoing detailed description is given merely byway of illustration and that many variations may be made therein withoutdeparting from the spirit of our invention.

Having described our invention, what we desire to secure by LettersPatent is:

1. A diagnostic preparation for the rapid and positive detection ofacetylmethylcarbinol which comprises a strip of bibulous materialimpregnated with:

(a) a medium zone capable of supporting the growth 5 ofEnterobacteriaceae;

(b) a reagent zone comprising:

(i) a member selected from the group consisting of:

l-arginine, the mono-, di, or tri acid addition salts of l-arginine,agmatine, and creatine; and (ii) an alkali metal salt ofa-naphthol-sulfonic acid, and

(c) a hydrophobic barrier zone separating said medium zone and saidreagent zone.

2. A diagnostic preparation according to claim 1 which has a secondhydrophobic barrier zone contiguous to said reagent zone, and on theopposite side of said reagent zone from said (0) barrier zone.

3. A diagnostic preparation according to claim 1 wherein said reagentzone comprises l-arginine monohydrochloride and an alkali metal salt ofoc-naphthol sulfonic acid.

4. A diagnostic preparation according to claim 3 wherein said reagentzone comprises 1.2 parts by weight of an alkali metal salt of u-naphtholsulfonic acid to 1 part of l-arginine monohydrochloride.

5. A diagnostic preparation according to claim 3 wherein said alkalimetal salt of naphthol sulfonic acid is l-naphthol-S-sulfonic acidsodium salt.

6. A diagnostic preparation according to claim 1 Wherein said mediumzone comprises peptone, dextrose and potassium phosphate.

7. A diagnostic preparation according to claim 6 wherein the ingredientsof said medium zone are present in a ratio of 7 parts peptone-S partsdextrose-5 parts potassium phosphate.

40 8. A diagnostic preparation according to claim 6 Wherein the optionalingredients D-glucose and sodium pyruvate are also included in themedium zone.

9. A diagnostic preparation according to claim 8 wherein the optionalingredients in the medium zone are present in an amount of from about 5to about 25 parts by weight of sodium pyruvate and from about 20 toabout 25 parts by weight of D-glucose.

10. A diagnostic preparation according to claim 2 wherein thehydrophobic barrier zones comprise an acrylic resin coating composition.

11. A process for the detection of acetylmethylcarbinol produced bymicro-organisms which comprises allowing the medium zone of a test stripas defined in claim 1 to come in contact with a suspension of a pureculture of 5 an unknown bacterium for about 4 hours at 37 0., adding anaqueous solution of an alkali metal hydroxide to said bacterialsuspension and allowing the alkali hydroxide treated bacterialsuspension to come in contact with the reagent zone of said test strip,and observing the 6 reagent zone for color change.

References Cited UNITED STATES PATENTS 3,011,874 12/1961 Deutch 195-1035OTHER REFERENCES Difco Manual 9th edition, pp. 54 and 55 (1953).

ALVIN E. TANENHOLTZ, Primary Examiner.

1. A DIAGNOSTIC PREPARATION FOR THE RAPID AND POSITIVE DETECTION OFACETYLMETHYLCARBINOL WHICH COMPRISES A STRIP OF BIBULOUS MATERIALIMPREGNATED WITH: (A) A MEDIUM ZONE CAPABLE OF SUPPORTING THE GROWTH OFENTEROBACTERIACEAE; (B) A REAGENT ZONE COMPRISING: (I) A MEMBER SELECTEDFROM THE GROUP CONSISTING OF: 1-ARGININE, THE MONO-, DI, OR TRI ACIDADDITION SALTS OF 1-ARGININE, AGMATINE, AND CREATINE; AND